Spinach line SMB66-1082F

ABSTRACT

The invention provides seed and plants of the spinach line designated SMB66-1082F. The invention thus relates to the plants, seeds and tissue cultures of spinach line SMB66-1082F, and to methods for producing a spinach plant produced by crossing a plant of spinach line SMB66-1082F with itself or with another spinach plant, such as a plant of another line. The invention further relates to seeds and plants produced by such crossing. The invention further relates to parts of a plant of spinach line SMB66-1082F, including the fruit and gametes of such plants.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding and, morespecifically, to the development of spinach line SMB66-1082F.

BACKGROUND OF THE INVENTION

The goal of vegetable breeding is to combine various desirable traits ina single variety/hybrid. Such desirable traits may include greateryield, resistance to insects or pests, tolerance to heat and drought,better agronomic quality, higher nutritional value, growth rate andfruit properties.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant or plant variety. A plant cross-pollinates if pollencomes to it from a flower of a different plant variety.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants of different varieties produces auniform population of hybrid plants that are heterozygous for many geneloci. Conversely, a cross of two plants each heterozygous at a number ofloci produces a population of hybrid plants that differ genetically andare not uniform. The resulting non-uniformity makes performanceunpredictable.

The development of uniform varieties requires the development ofhomozygous inbred plants, the crossing of these inbred plants, and theevaluation of the crosses. Pedigree breeding and recurrent selection areexamples of breeding methods that have been used to develop inbredplants from breeding populations. Those breeding methods combine thegenetic backgrounds from two or more plants or various other broad-basedsources into breeding pools from which new lines are developed byselfing and selection of desired phenotypes. The new lines are evaluatedto determine which of those have commercial potential.

One crop species which has been subject to such breeding programs and isof particular value is spinach. Spinach (Spinacia oleracea) is aflowering plant in the family Amaranthaceae native to central andsouthwestern Asia. Spinach is now grown in many temperate parts of theworld, but is most productive in cool seasons and climates as heat willcause spinach to bolt. It is an annual plant (rarely biennial) havingflowers that mature into a small hard dry lumpy fruit cluster about 5-10mm across containing several seeds.

Spinach has two stages in its life cycle including the vegetative,rosette stage in which the plant is marketable (about 35-40 days) andthe bolting, seed stalk stage in which the plant is no longermarketable. Spinach can grow in a range of soils as long as they aremoist and fertile, and particularly sandy loams that are high in organicmatter. However, the plants typically do poorly in acid soil, whichshould be at least a pH of 6.0, having an optimum pH of 6.2-6.9.

Spinach performs poorly against weeds. For weed control, usually 2-4cultivations are conducted in spinach fields, but fields should beshallow as to not harm any of the roots. Spinach also has a shallow rootsystem, so spinach grows best in uniformly moist conditions. Fields areirrigated by either flooding, furrow, or overhead sprinklers, furrow orflooding irrigation is preferred because overhead irrigation can reduceyields by increasing the risk and levels of disease that thrive inmoist, humid conditions.

Spinach is traditionally classified by sowing time (spring, summer, andwinter spinach) and harvesting method. Spinach is considered to bedioecious, although there is a continuous range of monoecious typesregarding the proportion of pistillate to staminate flowers per plant.In 1954, Janick and Stevenson (1954) reported a study of progenysegregation from selected crosses involving pistillate, staminate, andmonoecious types to clarify the genetic mechanisms that bring about themonoecious complex in spinach. Sex determination in dioecious strains ofspinach is controlled by a mechanism that acts as if it were a singlefactor pair; the pistillate plant is homozygous (XX), and the staminateplant is heterozygous (XY). The monoecious character in spinach appearsto be controlled by one major gene, X^(m), which was found to be allelicto the X Y factor pair. X^(m) is incompletely dominant to X. The Yallele is dominant to X and X^(m).

While breeding efforts to date have provided a number of useful spinachlines with beneficial traits, there remains a great need in the art fornew lines with further improved traits. Such plants would benefitfarmers and consumers alike by improving crop yields and/or quality.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a spinach plant of theline designated SMB66-1082F. Also provided are spinach plants having allthe physiological and morphological characteristics of the spinach linedesignated SMB66-1082F. Parts of the spinach plant of the presentinvention are also provided, for example, including pollen, an ovule, afruit, and a cell of the plant.

The invention also concerns seed of spinach line SMB66-1082F. Thespinach seed of the invention may be provided as an essentiallyhomogeneous population of spinach seed of the line designatedSMB66-1082F. Essentially homogeneous populations of seed are generallyfree from substantial numbers of other seed. Therefore, seed of lineSMB66-1082F may be defined as forming at least about 97% of the totalseed, including at least about 98%, 99%, or more of the seed. Thepopulation of spinach seed may be particularly defined as beingessentially free from hybrid seed. The seed population may be separatelygrown to provide an essentially homogeneous population of spinach plantsdesignated SMB66-1082F.

In another aspect of the invention, a plant of spinach line SMB66-1082Fcomprising an added heritable trait is provided. The heritable trait maycomprise a genetic locus that is a dominant or recessive allele. In oneembodiment of the invention, a plant of spinach line SMB66-1082F isdefined as comprising a single locus conversion. In specific embodimentsof the invention, an added genetic locus confers one or more traits suchas, for example, herbicide tolerance, insect resistance, diseaseresistance, and modified carbohydrate metabolism. The trait may be, forexample, conferred by a naturally occurring gene introduced into thegenome of the line by backcrossing, a natural or induced mutation, or atransgene introduced through genetic transformation techniques into theplant or a progenitor of any previous generation thereof. Whenintroduced through transformation, a genetic locus may comprise one ormore transgenes integrated at a single chromosomal location.

In another aspect of the invention, a tissue culture of regenerablecells of a plant of line SMB66-1082F is provided. The tissue culturewill preferably be capable of regenerating plants capable of expressingall of the physiological and morphological characteristics of the line,and of regenerating plants having substantially the same genotype asother plants of the line. Examples of some of the physiological andmorphological characteristics of the line SMB66-1082F include thosetraits set forth in the tables herein. The regenerable cells in suchtissue cultures may be derived, for example, from embryos, meristems,cotyledons, pollen, leaves, anthers, roots, root tips, pistil, flower,seed and stalks. Still further, the present invention provides spinachplants regenerated from a tissue culture of the invention, the plantshaving all the physiological and morphological characteristics of lineSMB66-1082F.

In yet another aspect of the invention, processes are provided forproducing spinach seeds, plants and fruit, which processes generallycomprise crossing a first parent spinach plant with a second parentspinach plant, wherein at least one of the first or second parentspinach plants is a plant of the line designated SMB66-1082F. Theseprocesses may be further exemplified as processes for preparing hybridspinach seed or plants, wherein a first spinach plant is crossed with asecond spinach plant of a different, distinct line to provide a hybridthat has, as one of its parents, the spinach plant line SMB66-1082F. Inthese processes, crossing will result in the production of seed. Theseed production occurs regardless of whether the seed is collected ornot.

In one embodiment of the invention, the first step in “crossing”comprises planting seeds of a first and second parent spinach plant,often in proximity so that pollination will occur for example, mediatedby insect vectors. Alternatively, pollen can be transferred manually.Where the plant is self-pollinated, pollination may occur without theneed for direct human intervention other than plant cultivation.

A second step may comprise cultivating or growing the seeds of first andsecond parent spinach plants into plants that bear flowers. A third stepmay comprise preventing self-pollination of the plants, such as byemasculating the male portions of flowers, (i.e., treating ormanipulating the flowers to produce an emasculated parent spinachplant). Self-incompatibility systems may also be used in some hybridcrops for the same purpose. Self-incompatible plants still shed viablepollen and can pollinate plants of other varieties but are incapable ofpollinating themselves or other plants of the same line.

A fourth step for a hybrid cross may comprise cross-pollination betweenthe first and second parent spinach plants. Yet another step comprisesharvesting the seeds from at least one of the parent spinach plants. Theharvested seed can be grown to produce a spinach plant or hybrid spinachplant.

The present invention also provides the spinach seeds and plantsproduced by a process that comprises crossing a first parent spinachplant with a second parent spinach plant, wherein at least one of thefirst or second parent spinach plants is a plant of the line designatedSMB66-1082F. In one embodiment of the invention, spinach seed and plantsproduced by the process are first generation (F₁) hybrid spinach seedand plants produced by crossing a plant in accordance with the inventionwith another, distinct plant. The present invention further contemplatesplant parts of such an F₁ hybrid spinach plant, and methods of usethereof. Therefore, certain exemplary embodiments of the inventionprovide an F₁ hybrid spinach plant and seed thereof.

In still yet another aspect of the invention, the genetic complement ofthe spinach plant line designated SMB66-1082F is provided. The phrase“genetic complement” is used to refer to the aggregate of nucleotidesequences, the expression of which sequences defines the phenotype of,in the present case, a spinach plant, or a cell or tissue of that plant.A genetic complement thus represents the genetic makeup of a cell,tissue or plant, and a hybrid genetic complement represents the geneticmake up of a hybrid cell, tissue or plant. The invention thus providesspinach plant cells that have a genetic complement in accordance withthe spinach plant cells disclosed herein, and plants, seeds and plantscontaining such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.It is understood that line SMB66-1082F could be identified by any of themany well known techniques such as, for example, Simple Sequence LengthPolymorphisms (SSLPs) (Williams et al., 1990), Randomly AmplifiedPolymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF),Sequence Characterized Amplified Regions (SCARs), Arbitrary PrimedPolymerase Chain Reaction (AP-PCR), Amplified Fragment LengthPolymorphisms (AFLPs) (EP 534 858, specifically incorporated herein byreference in its entirety), and Single Nucleotide Polymorphisms (SNPs)(Wang et al., 1998).

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by spinach plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of a spinach plant of the invention with a haploid geneticcomplement of a second spinach plant, preferably, another, distinctspinach plant. In another aspect, the present invention provides aspinach plant regenerated from a tissue culture that comprises a hybridgenetic complement of this invention.

In still yet another aspect, the invention provides a plant of an inbredspinach line that exhibits a combination of traits comprising mid-latebolting, long petioles, open growth habit, dark green leaf color, curledup leaves at primary market stage, spineless seeds (smooth), a lowpercentage of flowers with anthers, stay-green characteristic understress conditions and a resistance to downy mildew (Peronospora farinosaf.sp. spinaciae (Pfs)) races Pfs 1 through Pfs 7. In certainembodiments, the combination of traits may be defined as controlled bygenetic means for the expression of the combination of traits found inspinach line SMB66-1082F.

In still yet another aspect, the invention provides a method ofdetermining the genotype of a plant of spinach line SMB66-1082Fcomprising detecting in the genome of the plant at least a firstpolymorphism. The method may, in certain embodiments, comprise detectinga plurality of polymorphisms in the genome of the plant. The method mayfurther comprise storing the results of the step of detecting theplurality of polymorphisms on a computer readable medium. The inventionfurther provides a computer readable medium produced by such a method.

In still yet another aspect, the present invention provides a method ofproducing a plant derived from line SMB66-1082F, the method comprisingthe steps of: (a) preparing a progeny plant derived from lineSMB66-1082F, wherein said preparing comprises crossing a plant of theline SMB66-1082F with a second plant; and (b) crossing the progeny plantwith itself or a second plant to produce a seed of a progeny plant of asubsequent generation. In further embodiments, the method mayadditionally comprise: (c) growing a progeny plant of a subsequentgeneration from said seed of a progeny plant of a subsequent generationand crossing the progeny plant of a subsequent generation with itself ora second plant; and repeating the steps for an additional 3-10generations to produce a plant derived from line SMB66-1082F. The plantderived from line SMB66-1082F may be an inbred line, and theaforementioned repeated crossing steps may be defined as comprisingsufficient inbreeding to produce the inbred line. In the method, it maybe desirable to select particular plants resulting from step (c) forcontinued crossing according to steps (b) and (c). By selecting plantshaving one or more desirable traits, a plant derived from lineSMB66-1082F is obtained which possesses some of the desirable traits ofthe line as well as potentially other selected traits.

These and other features and advantages of this invention are describedin, or are apparent from, the following detailed description of variousexemplary embodiments of the devices and methods according to thisinvention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants,seeds and derivatives of spinach line SMB66-1082F. This line showsuniformity and stability within the limits of environmental influencefor the traits described hereinafter. Spinach line SMB66-1082F providessufficient seed yield. By crossing with a distinct second plant, uniformF1 hybrid progeny can be obtained.

Line SMB66-1082F exhibits a number of beneficial traits including fastgrowth, mid-late bolting, long petioles, very open growth habit, darkgreen leaf color, curled up leaves at primary market stage, spinelessseeds (smooth), a relatively low percentage of flowers with anthers, forexample, as found for publicly known line SSB66-17F, stay-greencharacteristic under stress conditions, field tenability and aresistance to downy mildew (Peronospora farinosa f.sp. spinaciae (Pfs))races Pfs 1 through Pfs 7. The development of the line can be summarizedas follows.

A. Origin and Breeding History of Spinach Line SMB66-1082F

Spinach line SMB66-1082F was developed by pedigree selection from aSeminis hybrid ‘Spitfire’. Spitfire is a publicly known line and is thecross between the Seminis female line MSA66-1031F and the male lineSMB66-1025M. Spitfire was registered at NAK-T on Sep. 29, 2005, undernumber 14411 (new number SPN364). The origin and selections that led tothe development of SMB66-1082F can be summarized as follows (S=Selfing,M=Mass selection):

Year 1 F1 generation grown from Spitfire. No selection. Year 1 F2generation grown. No selection. Year 2 F2.S1 generation grown. Selectedfor Pfs 7 resistance, color and tenability. Year 2 F2.S2 generationgrown. Selected for Pfs 7 resistance, yield and color. Year 4 F2.S3generation grown. Selected for color, yield and tenability. Year 5F2.S3.M1 generation grown. Selected for uniformity.

Observations during the Mass selection one year and the stock seedproduction in another year confirm that SMB66-1082F is uniform andstable. As is true with other spinach varieties, a small percentage ofoff-types can occur for almost any characteristics during the course ofrepeated multiplications. No variants are known to occur.

B. Physiological and Morphological Characteristics of Spinach LineSMB66-1082F

In accordance with one aspect of the present invention, there isprovided a plant having the physiological and morphologicalcharacteristics of spinach line SMB66-1082F. A description of thephysiological and morphological characteristics of spinach lineSMB66-1082F is presented in Table 1.

TABLE 1 Physiological and Morphological Characteristics of LineSMB66-1082F and a Selected Variety CHARACTERISTIC SMB66-1082F SpitfireSpecies Spinacia oleracea L. Spinacia oleracea L. Ploidy Diploid DiploidMaturity Growth Rate Slow (Norgreen) Medium (Long Standing Bloomsdale)Days from planting to prime 72 64 market stage Plant (Prime MarketStage) Habit Semi-erect (Long Standing Semi-erect (Long StandingBloomsdale) Bloomsdale) Size Medium Medium Spread (cm) 28 26 Height (cm)12 13 Seedling Cotyledon Width (mm)  5  5 Length (mm) 54 61 Tip PointedPointed Color Medium Green Medium Green Color Chart Name RHS Color ChartValue 143A 143A Leaf (First Foliage Leaves) Shape Ovate Ovate BaseV-shape V-shape Tip Round-pointed Round-pointed Margin Flat Flat UpperSurface Color Dark Green (Long Standing Dark Green (Long StandingBloomsdale) Bloomsdale) Color Chart Name RHS Color Chart Value 137B 137ALower Surface Color Lighter Lighter (Compared with upper surface) ColorChart Name RHS Color Chart Value 137C 137C Leaf (Prime Market Stage)Surface Smooth (Viroflay) Smooth (Viroflay) Shape Ovate Ovate BaseStraight Straight Tip Round-pointed Round-pointed Margin Curled UpCurled Under Upper Surface Color Dark Green (Standing Dark Green(Standing Bloomsdale) Bloomsdale) Color Chart Name RHS Color Chart Value139A 139A Lower Surface Color Lighter Lighter (Compared with uppersurface) Color Chart Name RHS Color Chart Value 137B N138B Luster GlossyGlossy Blade Size Medium (Virginia Savoy) Medium (Virginia Savoy) BladeLobing Not Lobed Not Lobed Petiole Color Medium Green Medium Green ColorChart Name RHS Color Chart Value 138B 138A Petiole Red PigmentationAbsent Absent Petiole Length to the Blade  8  8 (cm) Petiole LengthMedium Medium Petiole Diameter (mm)  4  4 Petiole Diameter Medium MediumSeed Stalk Development Start of Bolting (10% of Medium (Long StandingMedium (Long Standing plants) Bloomsdale) Bloomsdale) Height of Stalk(cm) 80 114  Leaves on Stalk of Female Many Many Plant Leaves on Stalkof Male Many Many Plant Plants that are Female 91-100% 66-90% Plantsthat are Male 0% 0% Plants that are Monoecious 0-10% 11-35% Seed SurfaceSmooth Smooth Disease Reaction Downy Mildew Resistant Resistant(Peronospora spinaciae) Race 1 Downy Mildew Resistant Resistant(Peronospora spinaciae) Race 2 Downy Mildew Resistant Resistant(Peronospora spinaciae) Race 3 Downy Mildew Resistant (Peronosporaspinaciae) Race 4 Downy Mildew Resistant Not Tested (Peronosporaspinaciae) Race 5 Downy Mildew Resistant Not Tested (Peronosporaspinaciae) Race 6 Downy Mildew Resistant Not Tested (Peronosporaspinaciae) Race 7 Cucumber Mosaic Virus Susceptible Susceptible *Theseare typical values. Values may vary due to environment. Other valuesthat are substantially equivalent are also within the scope of theinvention.

Line SMB66-1082F has been self-pollinated and planted for a number ofgenerations to produce the homozygosity and phenotypic stability to makethis line useful in commercial seed production. No variant traits havebeen observed or are expected for this line.

Spinach line SMB66-1082F, being substantially homozygous, can bereproduced by planting seeds of the line, growing the resulting spinachplant under self-pollinating or sib-pollinating conditions andharvesting the resulting seeds using techniques familiar to one of skillin the art.

C. Breeding Spinach Line SMB66-1082F

One aspect of the current invention concerns methods for crossing thespinach line SMB66-1082F with itself or a second plant and the seeds andplants produced by such methods. These methods can be used forpropagation of line SMB66-1082F, or can be used to produce hybridspinach seeds and the plants grown therefrom. Hybrid seeds are producedby crossing line SMB66-1082F with second spinach parent line. LineSMB66-1082F can be used as either a male or female parent to produce ahybrid. In one embodiment, line SMB66-1082F is used as a female parent.

The development of new varieties using one or more starting varieties iswell known in the art. In accordance with the invention, novel varietiesmay be created by crossing line SMB66-1082F followed by multiplegenerations of breeding according to such well known methods. Newvarieties may be created by crossing with any second plant. In selectingsuch a second plant to cross for the purpose of developing novel lines,it may be desired to choose those plants which either themselves exhibitone or more selected desirable characteristics or which exhibit thedesired characteristic(s) in progeny. Once initial crosses have beenmade, inbreeding and selection take place to produce new varieties. Fordevelopment of a uniform line, often five or more generations of selfingand selection are involved.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny are heterozygous for locicontrolling the characteristic being transferred, but are like thesuperior parent for most or almost all other loci. The last backcrossgeneration would be selfed to give pure breeding progeny for the traitbeing transferred.

The line of the present invention is particularly well suited for thedevelopment of new lines based on the elite nature of the geneticbackground of the line. In selecting a second plant to cross withSMB66-1082F for the purpose of developing novel spinach lines, it willtypically be preferred to choose those plants which either themselvesexhibit one or more selected desirable characteristics or which exhibitthe desired characteristic(s) when in hybrid combination. Examples ofdesirable characteristics may include, but are not limited to,resistance to one or more viruses (such as cucumber mosaic virus, Beetwestern yellows virus, and Beet curly top virus, for example), one ormore fungi (such as white rust, blue mold, downy mildew, Fusarium wilt,damping off disease caused by Pythium, and sclerotinia, for example),one or more bacteria (such as corky root rot and Bacterial Soft rot, forexample), and one or more insects (green peach aphid, spinach leafminer(Pegomyia hyoscyami), Budworm injury (Hylemya cilicrura), cucumberbeetle, silverleaf whitefly, Cabbage looper, flea beetles, root maggotsand cutworms); or increased phenolic content to increase antioxidantcapacity (Howard et al., 2002), for example.

Non-limiting examples of genes that may be utilized for generatingtransgenic spinach include RAR1 disease resistance proteins, asdescribed in, for example, U.S. Pat. No. 7,098,378, the ability totolerate high salt conditions, as described in, for example, U.S. Pat.No. 7,041,875 or U.S. Pat. No. 6,936,750; trehalose synthase forincreased amounts of trehalose to increase tolerance to a variety ofstresses, in particular to decreased availability of water, as describedin, for example, U.S. Pat. No. 5,792,921; overexpression of phytochrome,such as for increased shade tolerance and/or darker green color, asdescribed in, for example, U.S. Pat. No. 5,268,526; expression ofreversibly glycosylated protein (RGP) for at least altered growth rates,as described in, for example, U.S. Pat. No. 6,194,638; improved growthunder low-light conditions, such as with COP1, as described in, forexample, U.S. Pat. No. 7,081,363 and so forth.

D. Performance Characteristics

As described above, line SMB66-1082F exhibits desirable agronomictraits, including mid-late bolting. This and other performancecharacteristics of the line were the subject of an objective analysis ofthe performance traits of the line relative to other lines. The resultsof the analysis are presented below.

TABLE 2 Performance Characteristics For Line SMB66-1082F Bolting (numberof days after sowing) Material Year 1 Year 2 Year 3 Height of Stalk (cm)Spitfire 66 56 61 114 SMB66-1082F 70 67 68 80

As shown above, line SMB66-1082F exhibits superior bolting related tocompeting lines. One important aspect of the invention thus providesseed of the variety for commercial use.

E. Further Embodiments of the Invention

When the term spinach line SMB66-1082F is used in the context of thepresent invention, this also includes plants modified to include atleast a first desired heritable trait. Such plants may, in oneembodiment, be developed by a plant breeding technique calledbackcrossing, wherein essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition toa genetic locus transferred into the plant via the backcrossingtechnique. The term single locus converted plant as used herein refersto those spinach plants which are developed by a plant breedingtechnique called backcrossing, wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to the single locus transferred into the varietyvia the backcrossing technique.

Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the present variety. The parentalspinach plant which contributes the locus for the desired characteristicis termed the nonrecurrent or donor parent. This terminology refers tothe fact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental spinach plant towhich the locus or loci from the nonrecurrent parent are transferred isknown as the recurrent parent as it is used for several rounds in thebackcrossing protocol.

In a typical backcross protocol, the original variety of interest(recurrent parent) is crossed to a second variety (nonrecurrent parent)that carries the single locus of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a spinach plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred locus fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single locus of the recurrent variety ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

In one embodiment, progeny diploid spinach plants of a backcross inwhich SMB66-1082F is the recurrent parent comprise (i) the desired traitfrom the non-recurrent parent and (ii) all of the physiological andmorphological characteristics of diploid spinach line SMB66-1082F asdetermined at the 5% significance level when grown in the sameenvironmental conditions.

Spinach varieties can also be developed from more than two parents. Thetechnique, known as modified backcrossing, uses different recurrentparents during the backcrossing. Modified backcrossing may be used toreplace the original recurrent parent with a variety having certain moredesirable characteristics or multiple parents may be used to obtaindifferent desirable characteristics from each.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,male sterility, herbicide resistance, resistance to bacterial, fungal,or viral disease, insect resistance, restoration of male fertility,modified fatty acid or carbohydrate metabolism, and enhanced nutritionalquality. These comprise genes generally inherited through the nucleus.

Direct selection may be applied where the single locus acts as adominant trait. An example of a dominant trait is the anthracnoseresistance trait. For this selection process, the progeny of the initialcross are sprayed with anthracnose spores prior to the backcrossing. Thespraying eliminates any plants which do not have the desired anthracnoseresistance characteristic, and only those plants which have theanthracnose resistance gene are used in the subsequent backcross. Thisprocess is then repeated for all additional backcross generations.

Selection of spinach plants for breeding is not necessarily dependent onthe phenotype of a plant and instead can be based on geneticinvestigations. For example, one can utilize a suitable genetic markerwhich is closely genetically linked to a trait of interest. One of thesemarkers can be used to identify the presence or absence of a trait inthe offspring of a particular cross, and can be used in selection ofprogeny for continued breeding. This technique is commonly referred toas marker assisted selection. Any other type of genetic marker or otherassay which is able to identify the relative presence or absence of atrait of interest in a plant can also be useful for breeding purposes.Procedures for marker assisted selection applicable to the breeding ofspinach are well known in the art. Such methods will be of particularutility in the case of recessive traits and variable phenotypes, orwhere conventional assays may be more expensive, time consuming orotherwise disadvantageous. Types of genetic markers which could be usedin accordance with the invention include, but are not necessarilylimited to, Simple Sequence Length Polymorphisms (SSLPs) (Williams etal., 1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR),Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858,specifically incorporated herein by reference in its entirety), andSingle Nucleotide Polymorphisms (SNPs) (Wang et al., 1998).

F. Plants Derived From Spinach Line SMB66-1082F by Genetic Engineering

Many useful traits that can be introduced by backcrossing, as well asdirectly into a plant, are those which are introduced by genetictransformation techniques. Genetic transformation may therefore be usedto insert a selected transgene into the spinach line of the invention ormay, alternatively, be used for the preparation of transgenes which canbe introduced by backcrossing. Methods for the transformation of plants,including spinach, are well known to those of skill in the art.Techniques which may be employed for the genetic transformation ofspinach include, but are not limited to, electroporation,microprojectile bombardment, Agrobacterium-mediated transformation anddirect DNA uptake by protoplasts.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner.

Production of transgenic spinach plants, including at least Spinaciaoleracea L., have been established. An exemplary protocol fortransforming transgenic spinach is described by Zhang and Zeevaart(1999), in which cotyledon explants were infected with Agrobacteriumtumefaciens strain LBA4404 carrying an exemplary selectable marker geneand reporter gene.

A particularly efficient method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesare coated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded are positioned at an appropriatedistance below the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a surfacecovered with target spinach cells. The screen disperses the particles sothat they are not delivered to the recipient cells in large aggregates.It is believed that a screen intervening between the projectileapparatus and the cells to be bombarded reduces the size of projectilesaggregate and may contribute to a higher frequency of transformation byreducing the damage inflicted on the recipient cells by projectiles thatare too large.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. ModernAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations(Klee et al., 1985). Moreover, recent technological advances in vectorsfor Agrobacterium-mediated gene transfer have improved the arrangementof genes and restriction sites in the vectors to facilitate theconstruction of vectors capable of expressing various polypeptide codinggenes. The vectors described have convenient multi-linker regionsflanked by a promoter and a polyadenylation site for direct expressionof inserted polypeptide coding genes. Additionally, Agrobacteriumcontaining both armed and disarmed Ti genes can be used fortransformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., 1985; U.S. Pat. No. 5,563,055).

Transformation of plant protoplasts also can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Omirulleh et al., 1993; Fromm et al., 1986;Uchimiya et al., 1986; Marcotte et al., 1988). Transformation of plantsand expression of foreign genetic elements is exemplified in Choi et al.(1994), and Ellul et al. (2003).

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for spinach plant geneexpression include, but are not limited to, the cauliflower mosaic virus(CaMV) P-35S promoter, which confers constitutive, high-level expressionin most plant tissues (see, e.g., Odel et al., 1985), including monocots(see, e.g., Dekeyser et al., 1990; Terada and Shimamoto, 1990); atandemly duplicated version of the CaMV 35S promoter, the enhanced 35Spromoter (P-e35S) the nopaline synthase promoter (An et al., 1988), theoctopine synthase promoter (Fromm et al., 1989); and the figwort mosaicvirus (P-FMV) promoter as described in U.S. Pat. No. 5,378,619 and anenhanced version of the FMV promoter (P-eFMV) where the promotersequence of P-FMV is duplicated in tandem, the cauliflower mosaic virus19S promoter, a sugarcane bacilliform virus promoter, a commelina yellowmottle virus promoter, and other plant DNA virus promoters known toexpress in plant cells.

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals can beused for expression of an operably linked gene in plant cells, includingpromoters regulated by (1) heat (Callis et al., 1988), (2) light (e.g.,pea rbcS-3A promoter, Kuhlemeier et al., 1989; maize rbcS promoter,Schaffner and Sheen, 1991; or chlorophyll a/b-binding protein promoter,Simpson et al., 1985), (3) hormones, such as abscisic acid (Marcotte etal., 1989), (4) wounding (e.g., wunl, Siebertz et al., 1989); or (5)chemicals such as methyl jasmonate, salicylic acid, or Safener. It mayalso be advantageous to employ organ-specific promoters (e.g., Roshal etal., 1987; Schernthaner et al., 1988; Bustos et al., 1989).

Exemplary nucleic acids which may be introduced to the spinach lines ofthis invention include, for example, DNA sequences or genes from anotherspecies, or even genes or sequences which originate with or are presentin the same species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA can include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a spinach plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a spinach plant include oneor more genes for insect tolerance, such as a Bacillus thuringiensis(B.t.) gene, pest tolerance such as genes for fungal disease control,herbicide tolerance such as genes conferring glyphosate tolerance, andgenes for quality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s). Forexample, structural genes would include any gene that confers insecttolerance including but not limited to a Bacillus insect control proteingene as described in WO 99/31248, herein incorporated by reference inits entirety, U.S. Pat. No. 5,689,052, herein incorporated by referencein its entirety, U.S. Pat. Nos. 5,500,365 and 5,880275, hereinincorporated by reference it their entirety. In another embodiment, thestructural gene can confer tolerance to the herbicide glyphosate asconferred by genes including, but not limited to Agrobacterium strainCP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat.No. 5,633,435, herein incorporated by reference in its entirety, orglyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No.5,463,175, herein incorporated by reference in its entirety.

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., 1991). The RNA could also be a catalytic RNA molecule (i.e., aribozyme) engineered to cleave a desired endogenous mRNA product (seefor example, Gibson and Shillito, 1997). Thus, any gene which produces aprotein or mRNA which expresses a phenotype or morphology change ofinterest is useful for the practice of the present invention.

G. Spinach End Uses and Products

Spinach has many well known end uses, any one of which may be used inconnection with a plant of the invention. Spinach is marketed toconsumers in forms that include fresh or frozen single vegetables ormixtures of vegetables, and components of various pre-prepared and/orpre-packaged items. Common examples include, but are not limited to,pre-washed, bagged spinach marketed for use in salads, alone or mixedwith other greens. Spinach may also be provided to consumers in formsthat can be conveniently stored, such as frozen or cannedsingle-servings, single- or mixed-vegetable packages designed for use incooking, and others. Spinach has a high nutritional value and thus theplants and plant parts provided by the invention will also findbeneficial use as an ingredient in various food products.

Therefore, in certain aspects of the invention, a food products isprovided comprising a plant according to the invention or any partthereof. Methods for the production of such products are also provided.In specific embodiments of the invention, a product is provided or amethod for the manufacture thereof, wherein the product is defined ascomprising leaf tissue from a plant provided herein.

H. Definitions

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

A: When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more.”

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor conferring malesterility or a chemical agent.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration: The development of a plant from tissue culture.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing, wherein essentially allof the desired morphological and physiological characteristics of aspinach variety are recovered in addition to the characteristics of thesingle locus transferred into the variety via the backcrossing techniqueand/or by genetic transformation.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Tetraploid: A cell or organism having four sets of chromosomes.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a spinach plant by transformation.

Triploid: A cell or organism having three sets of chromosomes.

I. Deposit Information

A deposit of spinach line SMB66-1082F, disclosed above and recited inthe claims, has been made with the American Type Culture Collection(ATCC), 10801 University Blvd., Manassas, Va. 20110-2209. The date ofdeposit was May 5, 2008. Upon issuance of a patent, all restrictionsupon the deposit will be removed, and the deposit is intended to meetall of the requirements of 37 C.F.R. §1.801-1.809. The accession numberfor those deposited seeds of spinach line SMB66-1082F is ATCC AccessionNo. PTA-9188. The deposit will be maintained in the depository for aperiod of 30 years, or 5 years after the last request, or for theeffective life of the patent, whichever is longer, and will be replacedif necessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

All references cited herein are hereby expressly incorporated herein byreference.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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1. A seed of spinach line SMB66-1082F a sample of seed of said linehaving been deposited under ATCC Accession Number PTA-9188.
 2. A plantgrown from the seed of claim
 1. 3. A plant part of the plant of claim 2.4. The plant part of claim 3, wherein said part is selected from thegroup consisting of a seed, pollen, an ovule, a leaf, and a cell.
 5. Aspinach plant, or a part thereof, having all the physiological andmorphological characteristics of the spinach plant of claim
 2. 6. Atissue culture of regenerable cells of spinach line SMB66-1082F, asample of seed of said line having been deposited under ATCC AccessionNumber PTA-9188.
 7. The tissue culture according to claim 6, comprisingcells or protoplasts from a plant part selected from the groupconsisting of embryos, meristems, cotyledons, pollen, leaves, anthers,roots, root tips, shoots, pistil, flower, seed and stalks.
 8. A spinachplant regenerated from the tissue culture of claim 6, wherein theregenerated plant expresses all of the physiological and morphologicalcharacteristics of spinach line SMB66-1082F, a sample of seed of saidline having been deposited under ATCC Accession Number PTA-9188.
 9. Amethod of producing seed, comprising crossing the plant of claim 2 withitself or a second spinach plant and allowing seed to form.
 10. Themethod of claim 9, comprising crossing the plant of spinach lineSMB66-1082F with a spinach plant of a different genotype relative tosaid line.
 11. An F1 hybrid seed produced by the method of claim
 10. 12.An F1 hybrid plant produced by growing the seed of claim
 11. 13. Amethod for producing a seed of a line SMB66-1082F-derived spinach plantcomprising the steps of: (a) crossing a spinach plant of lineSMB66-1082F, a sample of seed of said line having been deposited underATCC Accession Number PTA-9188, with a second spinach plant; and (b)allowing seed of a SMB66-1082F-derived spinach plant to form.
 14. Themethod of claim 13, further comprising the steps of: (c) crossing aplant grown from said SMB66-1082F-derived spinach seed with itself or asecond spinach plant to yield additional SMB66-1082F-derived spinachseed; (d) growing said additional SMB66-1082F-derived spinach seed ofstep (c) to yield additional SMB66-1082F-derived spinach plants; and (e)repeating the crossing and growing steps of (c) and (d) to generatefurther SMB66-1082F-derived spinach plants.
 15. A method of vegetativelypropagating a plant of spinach line SMB66-1082F comprising the steps of:(a) collecting tissue capable of being propagated from a plant ofspinach line SMB66-1082F, a sample of seed of said line having beendeposited under ATCC Accession Number PTA-9188; (b) cultivating saidtissue to obtain proliferated shoots; and (c) rooting said proliferatedshoots to obtain rooted plantlets.
 16. The method of claim 15, furthercomprising growing plants from said rooted plantlets.
 17. A method ofintroducing a desired trait into spinach line SMB66-1082F comprising:(a) crossing a plant of line SMB66-1082F, a sample of seed of said linehaving been deposited under ATCC Accession Number PTA-9188, with asecond spinach plant that comprises a desired trait to produce F1progeny; (b) selecting an F1 progeny that comprises the desired trait;(c) crossing the selected F1 progeny with a plant of line SMB66-1082F toproduce backcross progeny; (d) selecting backcross progeny comprisingthe desired trait and the physiological and morphological characteristicof spinach line SMB66-1082F; and (e) repeating steps (c) and (d) threeor more times in succession to produce selected fourth or higherbackcross progeny that comprise the desired trait.
 18. A spinach plantproduced by the method of claim 17, wherein the spinach plant comprisesessentially all of the physiological and morphological characteristicsof spinach line SMB66-1082F, a sample of seed of said line having beendeposited under ATCC Accession Number PTA-9188.
 19. A method ofproducing a plant of spinach line SMB66-1082F, a sample of seed of saidline having been deposited under ATCC Accession Number PTA-9188,comprising an added desired trait, the method comprising introducing atransgene conferring the desired trait into a plant of spinach lineSMB66-1082F to produce a plant of spinach line SMB66-1082F comprising anadded desired trait.
 20. A progeny plant of the plant of claim 2 thatcomprises all of the physiological and morphological characteristics ofspinach line SMB66-1082F, a sample of seed of said line having beendeposited under ATCC Accession Number PTA-9188.
 21. A seed that producesthe plant of claim
 20. 22. A method of determining the genotype of theplant of claim 2, comprising obtaining a sample of nucleic acids fromsaid plant and detecting in said nucleic acids a plurality ofpolymorphisms.
 23. The method of claim 22, further comprising the stepof storing the results of the step of detecting the plurality ofpolymorphisms on a computer readable medium.
 24. A method of producingspinach comprising: (a) obtaining the plant of claim 2, and (b)collecting leaf tissue from the plant.